Identification and Functional Investigation of SOX4 as a Novel Gene Underpinning Familial Atrial Fibrillation

Abstract

Background: Atrial fibrillation (AF) signifies the most prevalent supraventricular arrhythmia in humans and may lead to cerebral stroke, cardiac failure, and even premature demise. Aggregating strong evidence points to genetic components as a cornerstone in the etiopathogenesis of familial AF. However, the genetic determinants for AF in most patients remain elusive. Methods: A 4-generation pedigree with idiopathic AF and another cohort of 196 unrelated patients with idiopathic AF as well as 278 unrelated healthy volunteers were recruited from the Chinese population of Han ethnicity. A family-based whole-exome sequencing examination followed by a Sanger sequencing assay in all research subjects was implemented. The functional impacts of the identified SOX4 mutations were explored via a dual-reporter assay. Results: Two new heterozygous SOX4 mutations, NM_003107.3: c.211C>T; p.(Gln71*) and NM_003107.3: c.290G>A; p.(Trp97*), were observed in the family and 1 of 196 patients with idiopathic AF, respectively. The two mutations were absent in the 278 control individuals. The biochemical measurements revealed that both Gln71*- and Trp97*-mutant SOX4 failed to transactivate GJA1 (Cx43). Moreover, the two mutations nullified the synergistic activation of SCN5A by SOX4 and TBX5. Conclusions: The findings first indicate SOX4 as a gene predisposing to AF, providing a novel target for antenatal genetic screening, individualized prophylaxis, and precision treatment of AF.

Keywords: SOX4; atrial fibrillation; biochemical assay; human genetics; sequencing examination.

Figure 1

Pedigree suffering from atrial fibrillation. The pedigree was arbitrarily termed as Family AF-01. “+” signifies a member harboring the discovered heterogeneous SOX4 mutation; “–” represents a non-carrier of the discovered SOX4 mutation.

Figure 2

A representative electrocardiogram recorded from the proband (II-7) of Family AF-01. The standard 12-lead electrocardiogram illustrates atrial fibrillation.

Figure 3

A novel SOX4 mutation predisposing to atrial fibrillation. (A) Sequencing electropherograms exhibiting the heterogeneous SOX4 mutation identified in the index patient with atrial fibrillation (mutant) along with its wild-type control detected in an unaffected subject (wild type). An arrow pinpoints where the mutation occurs. (B) Schematic drawings describing the main structural domains of SOX4. TAD: transcriptional activation domain; HMG: high mobility group.

Figure 4

A novel SOX4 mutation contributing to atrial fibrillation. (A) Sequencing electropherograms displaying the heterogeneous SOX4 mutation detected in one of the cohort patients with atrial fibrillation (mutant) and its wild-type control detected in a control individual (wild type). An arrow directs where the mutation occurs. (B) Schematic diagrams illustrating the main structural domains of SOX4. HMG: high mobility group; TAD: transcriptional activation domain.

Figure 5

A representative electrocardiogram from the patient harboring the SOX4 c.290G>A mutation. The electrocardiogram documents atrial fibrillation.

Figure 6

Functional failure of Gln71*- or Trp97*-mutant SOX4. In COS-7 cells cultivated in vitro, dual-reporter measurement of the transactivation of the GJA1/Cx43 promoter-driven firefly luciferase by wild-type SOX4 or Gln71*-mutant SOX4 (Gln71*) or Trp97*-mutant SOX4 (Trp97*), separately or in combination, unveiled that the Gln71* mutant (A) or Trp97* mutant (B) failed to transactivate GJA1/Cx43. Here “a” and “c” mark p < 0.001, and “b” and “d” indicate p < 0.01, when compared with wild-type SOX4 (400 ng).

Figure 7

No Synergistic Transactivation of SCN5A Between TBX5 and Gln71*- or Trp97*-Mutant SOX4. In cultured HEK293 cells, dual-reporter gene assessment of the synergistic transactivation of the SCN5A promoter by TBX5 and SOX4 demonstrated that the synergy was disrupted by the Gln71* (A) or Trp97* (B) mutation. Herein “e”, “f” and “g” denote p < 0.005, and “h” means p < 0.001 when compared with the corresponding wild-type counterparts.

Figure 8

Multiple SOX4-binding sites located within the promoter of GJA1/Cx43. Red color was used to mark the SOX4-binding sites; green color: the first exon mRNA; underlined bases in bold: primer sequences.

Figure 9

Multiple SOX4- and TBX5-binding sites mapped within the promoter of SCN5A. Red color was used to mark the SOX4-binding sites; blue color: the TBX5-binding sites; green color: the first exon mRNA; underlined bases in bold: primer sequences.